Feed formulations are required to provide animals essential nutrients critical to growth. However, crop plants are generally rendered food sources of poor nutritional quality because they contain low proportions of several amino acids which are essential for, but cannot be synthesized by, animals.
For many years researchers have attempted to improve the balance of essential amino acids in the proteins of important crops through breeding programs. As more becomes known about storage proteins and the expression of the genes which encode these proteins, and as transformation systems are developed for a greater variety of plants, molecular approaches for improving the nutritional quality of seed proteins can provide alternatives to the more conventional approaches. Thus, specific amino acid levels can be enhanced in a given crop via biotechnology.
One alternative method is to express a heterologous protein of favorable amino acid composition at levels sufficient to obviate food or feed supplementation. For example, a number of seed proteins rich in sulfur amino acids have been identified. A key to good expression of such proteins involves efficient expression cassettes with seed specific promoters. Not only must the gene-controlling regions direct the synthesis of high levels of mRNA, the mRNA must be translated into stable protein.
Among the essential amino acids needed for animal nutrition, often missing from crop plants, are methionine, threonine and lysine. Attempts to increase the levels of these free amino acids by breeding, mutant selection and/or changing the composition of the storage proteins accumulated in crop plants has met with minimal success. Usually, the expression of the transgenic storage protein was too low. The phaseolin-promoted Brazil nut 2S expression cassette is an example of an effective chimeric seed-specific gene. However, even though Brazil nut protein increases the amount of total methionine and bound methionine, thereby improving nutritional value, there appears to be a threshold limitation as to the total amount of methionine that is accumulated in the seeds. The seeds remain insufficient as sources of methionine.
An alternative to the enhancement of specific amino acid levels by altering the levels of proteins containing the desired amino acid is modification of amino acid biosynthesis. Recombinant DNA and gene transfer technologies have been applied to alter enzyme activity catalyzing key steps in the amino acid biosynthetic pathway. Glassman, U.S. Pat. No. 5,258,300; Galili, et al., European Patent Application No. 485970; (1992); incorporated herein in its entirety by reference. However, modification of the amino acid levels in seeds is not always correlated with changes in the level of proteins that incorporate those amino acids. Burrow, et al., Mol. Gen. Genet.; Vol. 241; pp. 431-439; (1993); incorporated herein in its entirety by reference. Although significant increases in free lysine levels in leaves have been obtained by selection for DHDPS mutants or by expressing the E. coli DHDPS in plants, it remains to be shown that these alterations can increase bound target amino acids, which represent some 90% or more of total amino acids. Thus, there is minimal impact on the nutritional value of seeds.
Based on the foregoing, there exists a need for methods of increasing the levels of the essential amino acids, threonine, methionine and lysine in seeds of plants.
It is therefore an object of the present invention to provide methods for genetically modifying plants to increase the levels of the essential amino acid threonine in the plants.
It is a further object of the present invention to provide seeds for food and/or feed with higher levels of the essential amino acid threonine than wild species of the same seeds.